PROJECT SUMMARY Spatial and temporal control of gene expression is crucial for the development of multicellular organisms. Improper gene expression leads to many abnormalities including developmental disorders and cancer. In addition to genetic information and chromatin structure, the three dimensional spatial organization of the eukaryotic genome within the nucleus significantly contributes to genome function. Critical to this organization is a developmentally essential architectural protein, CCCTC-binding factor (CTCF). CTCF exerts its insulator function by forming looping interactions between nonadjacent segments of DNA. CTCF binds to a highly conserved motif on DNA that is enriched at the borders of topologically associating domains (TADs), which are the building blocks of genome organization. Currently, we know neither how CTCF functions as a barrier between distinct chromatin domains, nor how it forms loops between distant loci. CTCF binding and function at many loci can be regulated in several ways; possibly through methylation of DNA, posttranslational modifications of CTCF, interactions with other proteins (i.e. cohesin), interactions with RNA, and multimerization of CTCF. Although CTCF constitutively binds to numerous loci across cell types, it can exert distinct functions depending on the cellular context. These variations cannot be explained by CTCF binding alone. CTCF can bind to the same regions in different cell types although it acts as a barrier in one cell type but not in the other. In our model system, a CTCF binding event within the HoxA cluster has no affect on transcription in mouse embryonic stem cells (mESCs), while it functions as a chromatin insulator upon differentiation into motor neurons (MNs). This suggests that CTCF binding is necessary, but not sufficient to mediate its insulation function. Rather, downstream regulatory events must be required to achieve specificity. These events could include modification to CTCF or association with secondary proteins, among other possibilities. Therefore, we hypothesize that CTCF interacts with other factors to function as an insulator. Here, we describe genetic and biochemical screens to identify factors that affect the ability of CTCF to perform its insulator function. Both approaches can independently reveal crucial factors for insulation function, which will be further analyzed to explain their mechanism of action. Ultimately, the proposed project will have an impact on gene regulation, with the results being fundamentally important for developmental and diseased processes such as developmental defects (i.e. human limb malformations), neurological disorders, and cancer.